Fatty acids are a principal metabolic fuel for liver, kidney and heart. Oxidation of fatty acyl-CoA's to trans-2,3-enoyl CoA's in mitochondria is catalyzed by acyl-CoA dehydrogenases. The dehydrogenases are linked to the main respiratory chain via the electron transfer flavoprotein (ETF) and the ETF:ubiquinone reductase. Fatty acids can also be Beta-oxidized in liver and kidney peroxisomes. In these organelles, the oxidation of acyl-CoA's to enoyl CoA's is linked directly to reduction of O2, yielding H2O2. These proteins play a central role in lipid catabolism and an understanding of their activities is important in understanding the relationships between fatty acid oxidation and disease states resulting from perturbed fatty acid catabolism. These include uncontrolled diabetes and myocardial infarction. Qualitative or quantitative alterations of ETF and ETF:ubiquinone reductase are involved in some inherited acidemias. The aims of this research are (a) to further elucidate the catalytic mechanism(s) of the mitochondrial acyl-CoA dehydrogenases and a peroxisomal acyl-CoA oxidase; and to test recent hypotheses concerning the structure of intermediates and timing of proton and hydride transfer; (b) to compare and contrast the mechanistic details of the fatty acyl-CoA oxidase and dehydrogenase in an attempt to explain what causes the difference in activity of these two enzymes which catalize oxidation of the same substrate; this includes finding an explanation for the observed difference in product inhibition of these two enzymes; and (c) to correlate the observed differences in function with structural features elucidated by resonance Raman spectroscopy. The experiments involve the use of rapid kinetic techniques, resonance Raman spectroscopy, specific active site-directed inhibitors and chromophoric pseudo substrates to study the dehydrogenation of acyl-CoA substrates by two acyl-CoA dehydrogenases and an acyl-CoA oxidase. Our overall goal is to elucidate the chemistry of acyl-CoA oxidation and understand the factors which can modulate the activities of the redox proteins that participate in these reactions.